This is a proposal to investigate the molecular mechanisms underlying tumor cell invasion. Acquistion of invasive potential confers on tumor cells the ability to detach from the primary tumor and spread to distal sites. Using an invasive melanoma cell line as a model system, we have found that the small GTP-binding protein, ARF6, regulates cell invasion. In recent years, ARF6 has emerged as an important signaling molecule and has been shown to regulate endosome cycling, and actin remodeling in a variety of cell types, both of which can impinge on the acquisition of cell migratory/invasive potential. Our studies demonstrate that ARF6 activation is linked to the activation of ERK via a signaling pathway that appears to be essential for cell invasion. Silencing of ARF6 by siRNAs or expression of an ARF6 dominant negative mutant abolishes invasion in cell-based in vitro assays. We have also shown that activation of endogenous ARF6 increases in response to physiological stimuli that stimulate tumor cell invasion. In this application, we aim to investigate the mechanisms by which ARF6 regulates cell invasion. Based on preliminary findings, we hypothesize that ARF6 activation promotes tumor cell invasion at least in part, by regulating (1) the trafficking of metalloproteases and (2) cytoskeletal remodeling required for invadopodia formation. By using cell biological, molecular and biochemical approaches in both, cell and animal model systems, we will investigate the role of ARF6 in tumor cell invasion. We propose three specific aims. In aim 1, we will investigate the role of ARF6 in the regulation of protease traffic by examining its regulation of MT1-MMP traffic and the release of proteases from the cell surface. In aim 2, we will elucidate the signal transduction pathway linking ARF6 to Rac1 activation during cell invasion and ask if invadopodia formation is important for protease secretion. In aim 3, we will investigate the role of the ARF6 GTPase cycle in melanoma cell invasion in vivo using a mouse model system. We believe that the information gained from investigations will provide new insights into the regulation of protease trafficking, a relatively understudied area of cancer biology, and will broaden existing knowledge on the molecular changes that occur during tumor progression. It is hoped that these findings will ultimately aid in the design of new strategies for diagnostic and/or therapeutic intervention of cancer progression.